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Studies of telomeric single-stranded 3’overhangs in Naumovozyma castellii

Overå, Caroline (2017) MOBT01 20162
Degree Projects in Molecular Biology
Popular Abstract
The single-stranded telomere ends

The telomeres are tandem repeat DNA sequences at the ends of our chromosomes. They are very important for the protection of our genomic DNA, and keeps it from being degraded and helps it staying intact. Genomic DNA is usually double-stranded, but on the very ends of the telomeres we find single-stranded ends which are called the 3’overhangs. These overhangs are important as they are binding areas for protective proteins that keep telomeres from being damaged. In this thesis project, we investigated how long these 3’overhangs are in the budding yeast, Naumovozyma castellii, and the possible protection of the 3’overhangs by a telomere binding protein, Rap1.
The lengths of 3’overhangs

The... (More)
The single-stranded telomere ends

The telomeres are tandem repeat DNA sequences at the ends of our chromosomes. They are very important for the protection of our genomic DNA, and keeps it from being degraded and helps it staying intact. Genomic DNA is usually double-stranded, but on the very ends of the telomeres we find single-stranded ends which are called the 3’overhangs. These overhangs are important as they are binding areas for protective proteins that keep telomeres from being damaged. In this thesis project, we investigated how long these 3’overhangs are in the budding yeast, Naumovozyma castellii, and the possible protection of the 3’overhangs by a telomere binding protein, Rap1.
The lengths of 3’overhangs

The 3’overhangs come in many different lengths, from barely present at all, to long stretches. Sometimes, under certain conditions, these 3’overhangs even have preferred lengths. When investigating yeast cells grown to high densities it was uncovered a range of different lengths of the 3’overhangs and they seemed to be favouring a particular length of 70 nucleotides. Yeast cells were later forced to grow synchronously in S-phase (the cell-cycle phase when DNA is replicated), and the 3’overhangs had a very high amount of the 70-nucleotide overhangs. This has been confirmed to happen in N. castellii and a similar phenomenon has been observed in human cancer cells. We analyzed several different yeast strains in order to determine whether some strains are more prone to produce these 70-nucleotide 3’overhangs.

The Rap1 protein at the double-stranded telomere repeats
The budding yeast Rap1 protein binds to double-stranded DNA. We investigated whether the protein could protect the 3’overhangs if it is bound close to the single-stranded DNA. We also tested the part of the Rap1 protein that binds to the DNA, the DNA-binding domain (DBD), and shortened versions of the DBD. This would be able to tell us if the DBD seems to be of importance for protection. The Rap1 DBD was incubated with telomeric DNA sequences which made the protein bind to the DNA. This protein-DNA complex was then exposed to an enzyme which would digest single-stranded DNA. We could then see how much single-stranded DNA was digested, and if there was any difference between the original DBD and the shortened DBD variants.
Altogether, our experiments would be able to tell us more about how the telomeric 3’overhangs work and if the Rap1 protein offers any protection to them. Research into telomere biology is important to discover more about the mechanism of aging and cancer.

Advisor: Marita Cohn
Master´s Degree Project of 60 credits
Molecular Biology, Molecular Genetics and Biotechnology 2016-2017
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Overå, Caroline
supervisor
organization
course
MOBT01 20162
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
8925416
date added to LUP
2017-09-12 10:03:53
date last changed
2017-09-12 10:03:53
@misc{8925416,
  author       = {Overå, Caroline},
  language     = {eng},
  note         = {Student Paper},
  title        = {Studies of telomeric single-stranded 3’overhangs in Naumovozyma castellii},
  year         = {2017},
}